Amanda Hayward-Lester

Quantitative analysis of gene expression by ion-pair high-performance liquid chromatography

We have analyzed the utility of ion-pair reversed-phase HPLC for gene quantification by competitive reverse transcriptase polymerase chain reaction (RT-PCR). Competitive RT-PCR reactions employed various RNA competitors which shared high sequence similarity to the native transcripts for which they served as references. Competitive reactions resulted in the detection of two reaction products when reactions were analyzed by agarose gel electrophoresis, but three products when analyzed by HPLC. The third product was demonstrated to be a heteroduplex formed between mixed strands of native and competitor amplicons. Mathematical analysis of these competitive reactions indicated that identification and quantification of the heteroduplexes were essential to produce accurate gene quantification. PCR amplification efficiency was shown to be identical for native and competitor transcripts. However, RT efficiency differences were observed which may be sequence dependent. These differences were highly consistent across reactions for the same native and competitor inputs. Increasing the sequence similarity resulted in a competitor which had the same RT efficiency as the native transcript. Titration of various levels of competitor against native RNA resulted in the expected linear relationships which had slopes of unity. Quantitation could be performed with similar precision in single tube comparisons in which the initial abundance of the native transcript was calculated by knowledge of the final reaction product ratio and the initial competitor input level. The assay system is highly accurate, i.e. the measured level of gene expression reflected the actual copy number of the gene present in the sample. This was demonstrated by performing reactions in which known amounts of native transcript were quantified and the amount estimated by the assay was shown to be the same as the known amount added to the reaction. A similar approach has been devised for examining the relative levels of alternatively spliced isoforms. In this system, primers were selected to produce reaction products which served as their own internal competitors (by spanning the alternative splice site). Hormonal dependence of the ratio of abundance of two isoforms of the rabbit RUSH-1 gene was demonstrated.

Quantification of alternatively spliced RUSH mRNA isoforms by QRT-PCR and IP-RP-HPLC analysis: a new approach to measuring regulated splicing efficiency.

Quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and the ion-pair reverse-phase (IP-RP)-HPLC product purification and detection system were developed to facilitate the isolation and proportional quantification of alternatively spliced RUSH mRNAs. RUSH isoforms result from alternative splicing of a 57-bp exon and encode SNF/SWI-related proteins that bind to the uteroglobin promoter. QRT-PCR was performed using total RNA, and a pair of primers designed to flank the 57-bp exon. When more than one splice variant was expressed, IP-RP-HPLC identified the specific homoduplex products, as well as the heteroduplexes formed as a consequence of partial sequence complementarity between the products. Data analysis included the correct re-allocation of heteroduplex components to achieve accurate quantitation of changes in the relative levels of RUSH message isoforms. The preferential expression of the RUSH-1alpha isoform by all the tissues except estrous uterine endometrium and lactating mammary gland indicates RUSH pre-mRNAs are alternatively spliced in a tissue-specific manner. A 61-fold difference in the relative rate of RUSH pre-mRNA splicing is indicated by the difference in the ratios of RUSH mRNA isoforms from uterine endometrium and testis. Clearly, QRT-PCR and IP-RP-HPLC are powerful and versatile tools for the detection and quantitation of mRNA splice variants.

Quantification of Specific Nucleic Acids, Regulated RNA Processing, and Genomic Polymorphisms Using Reversed-Phase HPLC

The ability to make accurate quantitative measurements of specific nucleic acid molecules is becoming increasingly significant in biomedical science. One important clinical application is the quantification of viral nucleic acids in patients’ serum and tissues to assess viral burden for determination of disease course and efficacy of treatment. In this case, accuracy and precision of measurement are of primary importance, as decisions concerning selection of medication and dose may depend entirely on perceived change in viral load in an otherwise asymptomatic patient. A second utility in basic biomedical research is the quantification of specific mRNA molecules in functionally characterized cell types. These measurements are useful because they provide a prediction of protein abundance in cell samples in which proteins cannot be quantified directly. These studies often require microdissection to separate the tissues of interest from neighboring tissues. Again, accurate quantification is important for the effective comparison of the expression of multiple genes in multiple tissues and at different laboratories, often under conditions of physiological or pharmacological manipulation.

Renal Na+, K+-ATPASE In SHR: Studies of Activity And Gene Expression

The mechanism by which increased dietary intake of calcium reduces blood pressure in the spontaneously hypertensive rat is unknown. The present studies were designed to determine if there were alterations in the activity of the major membrane ion translocating pump, sodium, potassium-ATPase (NKA), in the kidneys of hypertensive rats and whether increased dietary calcium intake affected the activity of this enzyme. Fifteen-week old SHRs were found to have lower total ATPase activity in microsomal preparations from the kidney than age matched Wistar-Kyoto animals. Both the ouabain-sensitive component (NKA) and the ouabain-insensitive component were lower in SHR. Increasing dietary calcium intake from 1% to 3% elevated both components of the ATPase activity in SHR, but was without effect in WKY. Measurement of membrane phospholipid composition suggested that altered phospholipid composition did not account for the reduced ATPase activity observed, but indicated a reduced density of ATPase in SHR. A technique has been devised for qualitative and quantitative analysis of Na, K-ATPase alpha isoforms using RT-PCR. This technique reveals that the alpha 1 isoform is the sole catalytic isoform present in the nephron. Accurate and precise quantification of the amount of gene expression in individual nephron segments is reported and will be applied to determine whether dietary calcium influences blood pressure by a mechanism which alters nephron NKA gene expression.

Q-RT-PCR: data analysis software for measurement of gene expression by competitive RT-PCR

MOTIVATION We have developed software to assist in the computation of gene expression from data obtained in competitive reverse transcription-polymerase chain reaction (RT-PCR). This report describes the mathematical basis of competitive RT-PCR and discusses the criteria which must be met to permit accurate estimations of gene expression to be obtained using this technique. RESULTS The software that has been developed assists in both the assessment of assay performance (specifically in establishing the equality of amplification efficiency of the native and competitor templates) and in the routine analysis of data obtained in quantitation of gene expression by competitive RT-PCR. The software is a 100 kb module which functions as a Microsoft Excel add-in. It is compatible with both Windows and Mac versions of Excel 5 and Excel 7 on the Windows 95 platform, and employs the spreadsheet, statistical and graphing capabilities incorporated into Excel. AVAILABILITY The software can be downloaded from http://www.grad.ttuhsc.edu/archive/. A brief summary in both HTML and Microsoft Word 6 format of the installation and use of the software is also located at this website.